A major problem for transfusion therapy for chronic anemia is the high degree of genetic diversity in blood group antigens in people of Asian and African backgrounds compared to white Europeans, who are the majority of blood donors. These polymorphic antigens on RBCs contribute to the high incidence of allo-immunization and presence of multiple antibodies in the serum of these patients. Identifying the specificity of thes antibodies and determining if they are auto- or allo- antibodies is critical for providing compatibe blood. The major goal of this application is to take advantage of the progress that has been made in the in vitro production of cultured red blood cells (cRBCs) by expansion of hematopoietic stem cells and hematopoietic progenitor cells, coincident with the progress made in the genetic characterization of rare blood groups, to develop reagent cRBCs to identify the presence of clinically significant antibodies to high prevalence donor antigens. Reagent cRBCs will facilitate antibody identification in highly allo-immunized multiply transfused patients, and streamline and standardize testing. We have designed a panel of 6 donors with rare blood group phenotypes that are sufficient to resolve most antibody identification problems seen in patients who have auto- and allo-antibodies, and that could be used to transfuse patients who currently cannot be transfused because of shortage of appropriate rare blood. In Aim 1, we propose to develop new culture methods to expand hematopoietic cells collected from peripheral blood or differentiated from iPSCs by sequential amplification of the HSC, HPC and erythroblast compartments. We also propose to generate induce-pluripotent stem cells from these 6 donors and to engineer a group O, Rh null line of iPSCs that are useful as reagent red cells and that could be used as universal cells for transfusion. Expression of surface antigens on cRBCs produced from peripheral blood or from iPSCs has not been extensively characterized. In Aim 2, we propose to compare cRBCs to native red blood cells from the same donor to determine if they would be suitable as reagent cells and for transfusion. We will focus on the antigenic profiles, the genotypes, the storage stability, and the performance of cRBCs in traditional blood bank assays. Importantly, the reagent cRBCs that we will produce are the same cells that are in short supply for transfusion of allo-immunized patients. The method development and the quality testing that we propose to perform will therefore pave the way to the production of sufficient amounts of rare cRBCs for life saving transfusion therapies and define the first specific application, production of reagent red cells, and the first human indication, transfusion support for SCD patients without other options.